This project proposes to study the mechanisms of phagocytosis and bacterial killing in the human leukocyte. Emphasis will be placed on the oxidative metabolism of the cell in an attempt to discover the biochemical basis of the respiratory burst and the mechanisms involved in the oxygen-dependent bactericidal activity. The NBT-diaphorase activities of normal cells will be investigated using both NADH and NADPH as substrates. Total diaphorase activities will first be characterized in broken cell preparations in terms of kinetic parameters, etc., then the enzymes will be separated by disc gel electrophoresis. Kinetic analysis and characterization will be performed on the separate isoenzymes. Particular emphasis will be placed on the intracellular location of the isoenzymes and on changes in the enzymatic activity upon phagocytosis. The possible role of superoxide anion in the bactericidal event will be investigated. Bacteria containing varying levels of superoxide dismutase will be prepared by changing the composition of the growth media. The ability of PMNL to kill these strains of bacteria will be tested in an in vitro system. Other approaches will include an attempt to directly document O2 formation in phagocytizing PMNL by electron spin resonance measurements. The effect of drugs which generate superoxide (such as dialuric acid and phenazine methosulfate) on PMNL metabolism will be examined. The ability of the isolated diaphorases to generate O2 will be examined, using the SOD sensitive reduction of cytochrome C as an assay procedure. It is hoped that these approaches will elucidate the normal mechanisms of leukocytic bactericidal activity and suggest methods which might improve the bactericidal ability of leukocytes in patients with severe infection.